In vivo effects of allogeneic mesenchymal stem cells in a rat model of acute ischemic kidney injury

Objective(s): Renal ischemia-reperfusion injury (IRI) as a severe condition of acute kidney injury (AKI) is the most common clinical problem with high mortality rates of 35-60% deaths in hospital. Mesenchymal stem cells (MSC) due to unique regenerative characteristics are ideal candidates for the treatment of the ischemic injuries. This work is focused on the administration of MSC to IRI-induced AKI Wistar rats and evaluating their significance in AKI treatment. Material and Methods: Animals underwent surgical procedure and AKI was induced by 40 min bilateral renal pedicle clamping. Immediately after reperfusion, 2×106 rat bone marrow derived MSCs were injected via intra-parenchymal or intra-aortic route. Results: Animals subjected to AKI after days 1 and 3 showed significant increase in the serum creatinine and blood urea nitrogen (BUN) concentration along with a declined glomerular filtration rate (GFR) when compared with non-ischemic animals. On the other hand, treated animals showed a significant enhanced regeneration as compared to ischemic animals in both administration route groups. Conclusion: According to the results concluded from the renoprotective effects of MSC in IRI/AKI, MSCs could be considered as promising therapeutic approach for AKI in clinical applications.


Introduction
Acute kidney injury (AKI), a heterogeneous clinical syndrome, refers to the progressively kidney failure and declined glomerular filtration rate (GFR) resulting in the accumulation of urea and creatinine in the body. This accumulation of nitrogen wastes results in the impairment of body fluid, electrolytes, and acid-base homeostasis, which is accompanied by significant morbidity and mortality i.e. 23.9% among adults and 13.8% among children. The incidence of AKI among individuals, who have been hospitalized, is significantly greater than the general population (1, 2) and ischemiareperfusion injury (IRI) has been known as the most common cause of AKI (3). Early diagnosis of AKI is difficult causing irreversible kidney damages in patients, which is being treated with current applied approaches such as dialysis or transplantation (4,5). Several cell culturing and pre-clinical approaches in animal models are being used to understand the pathophysiology of AKI and finding novel and effective therapeutic strategies (2). Renal IRI is a common experimental model to induce AKI in rat and mice for experimental purposes (2). As per literature, following the induction of renal IRI, the inflammation process is activated and numerous inflammatory mediators such as cytokines, chemokines, and reactive oxygen species (ROS) are secreted and activated, causing recruitment of inflammatory cells for enhanced inflammation leading towards apoptosis and necrosis (4,6).
For these reasons, fundamental strategies and cures are needed for the management and treatment of AKI.
The significant damage of ischemic injury is evidenced in the corticomedullary zone, in particular, S3 segment of the proximal tubule, located in the deep inner cortex and the outer stripe of the outer medulla (2). In spite of the paracrine effects of MSCs, their efficiency to establish a natural cell niche and to regenerate the injury is directly related to the closeness of injury site and administration of the cells. The aim of the present study was to investigate the regenerative potential of MSCs administered immediately after reflow and to compare systemic and locally injection route, in a rat model of IRI/AKI.

Animals, induction of IRI/AKI, and cell infusions
All procedures involving animals were approved by the respective Institutional Animal Care of the Mashhad University of Medical Sciences (Mashhad, Iran) and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animals were housed at a constant temperature and humidity, with a 12:12-hr light-dark cycle, and had unrestricted access to a standard diet and tap water. Adult male Wistar rats weighing 250-300 g were used for this study (MUMS, Iran).
The corticomedullary zone is the best site for locally intraparenchymal injection, so in the absence of the ultrasonic guide, the injection at the proper location would be blind. We studied right and left kidneys of the 40 healthy (control) adult male Wistar rats that were killed in our labs for other experiments. Kidneys were removed and fixed in 10% formalin and dehydrated with different degrees of alcohol and paraffin-embedded blocks were prepared. Two sections (5-micrometer) were prepared from each block and were stained with hematoxylin and eosin (H&E) and the slides were evaluated by morphometric method. To confirm the location of injection site, methylene blue solution was injected in the corticomedullary zone, and then injection site was examined. Thirty gauge needles with length of 1 cm were used, while a micropipette tip of 7 mm was cut from the top and the needle was entered to it, so that 3 mm from the tip of the needle was available for injection.
The rats were randomly assigned to the following groups, each containing of five animals: sham-operated group, systemic/local vehicle treated groups (Sys/Locvehicle), IRI with systemic/local vehicle treated groups (Sys/Loc-IRI), and IRI with systemic/local cell treated groups (Sys/Loc-IRI+MSC).
IRI/AKI was induced in pentobarbital-anesthetized animals, and rectal temperature was maintained at 37°C, as described elsewhere (23). After a midabdominal laparotomy, kidneys were exposed and renal pedicles were clamped with atraumatic vascular clamps for 40 min. While clamps were applied, in systemic cell/ vehicle treated groups, the abdominal aorta was cannulated with IV cannula (gauge 24, O.D. 0.7 mm) for intra-aortic injection. In locally cell/vehicle-treated groups, intraparenchymal injection was performed, as described above. The vehicle in control rats with IRI was infused by the same route. The sham-operated animals underwent similar operative procedures but without any other procedure.
Administration of cells or vehicle was performed immediately after reflow. After visual confirmation of reflow, ~2×10 6 MSC/animal were given via the suprarenal aorta or intra-parenchymal of kidney (~1×10 6 MSC/kidney). Control animals for IRI were infused with medium. Aorta puncture site was sutured with 10-0 nylon, covered by fibrin glue and abdominal incision was closed with 4-0 silk, and animals were allowed to recover.

Cells and culture
MSCs used for this experiment were generated by standard procedures. Briefly, under sterile conditions 6-weeks old male Wistar rats were killed by chloroform. Then, the femur and tibia were excised and all connective tissues attached to bones were removed immediately (24). Bone marrow plugs were extracted by flushing the bone marrow cavity in sterile PBS, and then the suspension was filtered through 100-µm mesh. The cells were centrifuged, resuspended in complete culture medium containing low-glucose Dulbecco's modified Eagle's medium (LG-DMEM; Gipco, USA) supplemented with 10% fetal bovine serum (FBS; Gipco, USA), plated in 75-cm 2 primary culture flasks (Spl, Life science; Korea), and incubated at 37°C in humidified atmosphere with 5% CO 2 . After 3 days, the medium was changed and nonadherent cells were removed. The MSCs were isolated on the basis of its ability to adhere to the culture flask. At 90% confluence, the cells were trypsinized (0.25% trypsin-EDTA; Gipco, USA) and passaged at 1:3 ratios. The culture medium was changed every 2-3 days. Cells had a typical spindle-shaped appearance, and the MSCs phenotype was confirmed by differentiation into osteocytes and adipocytes with specific differentiation media (25). In addition, superficial markers were negative for CD45, CD11b, CD34 and positive for CD44, CD166, CD73 and CD90 expression, determined by Fluorescence-activated cell sorting (FACS) analysis (28). Passage 3-4 was used in all experiments. The viability of MSC by using trypan blue staining was verified to be >85% before use.

Biochemical analysis
Blood samples were collected at baseline and days 1 and 3 post-IRI. Urine samples were obtained from metabolic cage for 24 hr at baseline and days 1 and 3 post-IRI. Creatinine and blood urea nitrogen (BUN) concentration were determined by autoanalyzer (BT3000 Plus, Italy).

Histology and injury scores
Kidney tissues were embedded in paraffin and 5 µm tissue sections obtained for H&E staining. Histological damage was scored by the EGTI scoring system. The scoring system consists of histological damage in 4 individual components: Endothelial, Glomerular, Tubular, and Interstitial (score: 0-14). Ten non overlapping fields were randomly chosen from the cortical and corticomedullary junction areas of each section (400 ×magnification) (29).

Statistical analysis
Data are expressed as means±SEM. Primary data collection and statistical analyses were carried out using Prism (GraphPad, San Diego, CA). ANOVA and Bonferoni's post hoc tests were used to assess differences between data. A P-value of <0.05 was considered significant.

Results
Animals were used in this study had a normal blood and urine tests at pre-surgery stage. All animals in control, sham-operated groups and pre-injury day in the treated groups showed a normal kidney function evaluated by serum creatinine and BUN and GFR measurement as well as normal renal histology.

Bone marrow -MSCs isolation, culture and characterization
MSCs, used in the current study, were generated by standard procedures and grown for at least three passages in culture and then morphologically defined by a fibroblast-like appearance ( Figure 1). Before in vivo usage of MSCs, they were characterized by confirming their ability to undergo osteogenic and adipogenic differentiation ( Figure 2) as well as flowcytometry analysis. The cells were negative for CD45, CD11b, and CD34 but positive for the CD44, CD166, CD73 and CD90 superficial markers (Figure 3).

Intraparenchymal injection
Histology study of 40 healthy animals revealed that average diameter of kidney cortex in adult male Wistar rat was 2.8 mm (Mean±SEM (2.81±0.7)) as the intraparenchymal injection was performed in 3 mm deep. Due to the limited access to ultrasound guide, injection at the proper location would be blind and we tried our best for a convenient alternative way. While studying the right and left kidneys of 40 healthy (control groups) adult male Wistar rats, we observed

Biochemical analysis and functional study
There were no significant differences between shamoperated group, systemic and local vehicle treated groups in serum creatinine, BUN, and GFR values at any time (Table 1-3).
Ischemia for 40 min in animals led to severe renal insufficiency, as evidenced by a rise in creatinine, BUN, and decline in GFR values when compared with nonischemic animals at days 1 and 3.
As shown in Figures 4-6, animals subjected to AKI at 24 hr post-ischemia had significant increase in the levels of serum creatinine to 3.41±0.23 mg/dl (Sys-IRI), 3±0.14 mg/dl (Loc-IRI), and serum BUN to 86.8±6.7 mg/dl (Sys-IRI) and 83.5±4.7 mg/dl (Loc-IRI) when compared with non-ischemic animals (P<0.05). The results showed that there are no significant differences between systemic and local ischemic groups at any time ( Table 1-3).

Histological analysis
We examined the histological characteristics of kidney tissues from each group. Compared with the sham-operated and systemic/local vehicle-treated groups injected with media, there was notable damage in IRI kidneys (Figure 7). When compared with IRI rats, 11±0.5 (Sys-IRI) and 10±0.5 (Loc-IRI), kidneys from Table 1

. Effect of renal ischemia and mesenchymal stem cells (MSC) administration on serum creatinine level
Serum creatinine values (mg/dl) from cell-treated and non-treated groups. There is no significant difference between systemic and local MSC treated animals. Values are mean±SEM. °P<0.0001 compared to day 0 in same group, *P<0.0001 compared to Sys-IRI group, day 1 and, #P<0.0001 compared to Loc-IRI group, day 1, ≠P<0.0001, and …P=0.0002 compared to Sham-operated group in same day, and •P<0.0001, and §P=0.0003 compared to vehicle-treated group in same route injection and same day

Table 2. Effect of renal ischemia and mesenchymal stem cells (MSC) administration on serum blood urea nitrogen (BUN) level
Serum BUN values (mg/dl) from cell-treated and non-treated groups. Values are mean±SEM. °P<0.0001, and °°P=0.0002 compared to day 0 in same group, *P=0.002, and **P<0.0001 compared to Sys-IRI group, day 1 and, #P=0.0005, and ##P<0.0001 compared to Loc-IRI group, day 1, ≠P<0.0001, …P=0.0056, and -P=0.006 compared to Sham-operated group in same day, and •P<0.0001, §P=0.0037, and "P=0.0135 compared to vehicle-treated group in same route injection and same day Table 3. Effect of renal ischemia and mesenchymal stem cells (MSC) administration on glomerular filtration rate (GFR) level GFR values (ml/min/100 gBW) from cell-treated and non-treated groups. There is no significant difference between systemic and local MSC treated animals. Values are mean±SEM. °P<0.0001 compared to day 0 in same group, *P=0.0012, and **P=0.0002 compared to Sys-IRI group, day 1 and, #P=0.0012, and ##P=0.0004 compared to Loc-IRI group, day 1, ≠P<0.0001 compared to Sham-operated group in same day, and •P<0.0001, §P=0.0003, and "P=0.0002 compared to vehicletreated group in same route injection and same day  that average cortex diameter is about 2.8 mm and if the injection is down in 3 mm from outside of the kidney, the top of the needle will be placed in the corticomedullary zone.

Discussion
The present study provides evidence to support that the MSC therapy contributes significant renoprotection in rats with IRI-AKI. Animals infused with MSC immediately after reperfusion had significantly better renal function and lower renal injury scores than untreated animals. This was demonstrated in two different injection route.
The pathophysiological process after ischemic injury causes several functional and structural changes in renal tissue involving cellular metabolism, cytoskeleton, extra cellular matrix generation and activation of inflammatory factors such as cytokines, chemokines, and ROS molecules (30).
Despite these changes, surviving renal tubule cells have a remarkable ability to regenerate and proliferate after ischemic AKI. As our results showed, untreated animals had a relative recovery in renal function 72 hr after ischemia.
Cell-based regenerative therapeutic applications are being considered as the most promising approaches to treat several diseases including IRI/AKI (11,(31)(32)(33)(34). Cellular mechanisms such as transdifferentiation, de- The protective effects via differentiation-independent mechanisms, is due to cytokines secretion and increased expression of growth factors such as HGF, VEGF, and IGF-I, or their anti-apoptotic and mitogenic properties (3,4,22,(35)(36)(37)(38). Significance of these paracrine effects of MSCs in regenerative medicine has gained much attention of scientists for IV injection of cells. In spite of such paracrine effects, the best administration route for the most effectiveness of stem cells is to be close to the site of injury and natural cell niche. It has been shown that the effect of cell injection at the site of injury in some tissues is much better than systemic administration. Several studies have focused on the local injection of the cells close to the injury site in ischemic heart and brain i.e. in many myocardial infarction models, direct injection of stem cells at the center of infarct and border zone areas of the myocardium is more effective than systemic injection (11,(31)(32)(33)(34).
It is well known that renal ischemia usually damages the corticomedullary zone. The S3 segment represents the remainder of the proximal tubule, located in the deep inner cortex and the outer stripe of the outer medulla (2). Because of that, the corticomedullary zone is the best site for locally intraparenchymal injection. In one study, cells were injected by hydrogel to increase homing in the tissue (39). In other two studies, the cells were injected into the corticomedullary zone under ultrasound guide (40,41).
As mentioned, the corticomedullary zone is the best site for intraparenchymal injection, so in the absence of the ultrasonic guide, the injection at the proper location was blind. We studied the 80 healthy kidneys and achieved cortical diameter.
The integration percentage of administrated stem cells is usually below 1% in any given organ. On the other hand, MSCs are known to have immunomodulatory properties and secrete various growth factors and cytokines, which may explain further possible mechanisms including inhibition of fibrosis and apoptosis, improvement of angiogenesis, stimulation of mitosis, proliferation and differentiation of intrinsic stem cells (42)(43)(44)(45). The kidney can recover after damage following repolarization, cell de-differentiation, migration, and proliferation and the release of growth factors, chemokines and cytokines can facilitate repair process (30).
The administration of MSC, hematopoietic stem cell (HSC), or a bone marrow transplant, following glycerol-, cis-platinum, or IRI/AKI, protected kidney tissue and improved renal function in rodents (3,44). As with other studies (3,44), animals infused with MSC, either systemic or local injection, compared with untreated MSC animals, had significantly better renal function as early as 24 hr following reflow (3,4), which was determined by decreasing the Cr level approximately 33%.
In accordance with comparative analysis of literature, it was observed that administration of MSC, independently to the administration route, significantly improved renal function at days 1 and 3 after induction of IRI/AKI among animals treated by MSCs.
The mechanism behind the regeneration could be transdifferentiation of MSCs and differentiationindependent protective effects such as cytokines secretion and increased expression of growth factors such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor 1 (IGF-I), or their anti-apoptotic and mitogenic properties. Pathological data was enough to justify the paracrine effects in MSC-based kidney regeneration (3,4,22,(35)(36)(37)(38). As discussed by several other studies, the infusion of MSC into the suprarenal aorta in IRI/AKI model has no adverse effects like respiratory and renal problems or death (4,44). Moreover, the advantage of local injection is directly trapping into the injury site. Results of the current work demonstrate that local injection has slightly better effect than intra-aortic injection but these differences are not statistically significant.

Conclusion
Our data showed the promising kidney-protective effect of MSC in the rat IRI/AKI experimental model employing both intra-aorta and intra-parenchymal injection routes. Administration of MSCs significantly improved renal function and histopathologic indices at days 1 and 3 after induction of IRI/AKI. These effects at least in part, mediated by complex paracrine actions that are able to significantly protect and regenerate the damaged kidneys. More investigations are required to determine the mechanism responsible for this recovery.